Establishing embryonic territories in the context of Wnt signaling.

This review highlights the work that my research group has been developing, together with international collaborators, during the last decade. Since we were able to establish the Xenopus laevis experimental model in Brazil, we have been focused on understanding early embryonic patterns regarding neural induction and axes establishment. In this context, the Wnt pathway appears as a major player and has been much explored by us and other research groups. Here, we chose to review three published works which we consider to be landmarks within the course of our research and also within the history of modern findings regarding neural induction and patterning. We intend to show how our series of discoveries, when painted together, tells a story that covers crucial developmental windows of early differentiation paths of anterior neural tissue: 1. establishing the head organizer in contrast to the trunk organizer in the early gastrula; 2. deciding between neural ectoderm and epidermis ectoderm at the blastula/gastrula stages, and 3. the gathering of prechordal unique properties in the late gastrula/early neurula.

Depletion of Ric-8B leads to reduced mTORC2 activity.

mTOR, a serine/threonine protein kinase that is involved in a series of critical cellular processes, can be found in two functionally distinct complexes, mTORC1 and mTORC2. In contrast to mTORC1, little is known about the mechanisms that regulate mTORC2. Here we show that mTORC2 activity is reduced in mice with a hypomorphic mutation of the Ric-8B gene. Ric-8B is a highly conserved protein that acts as a non-canonical guanine nucleotide exchange factor (GEF) for heterotrimeric Gαs/olf type subunits. We found that Ric-8B hypomorph embryos are smaller than their wild type littermates, fail to close the neural tube in the cephalic region and die during mid-embryogenesis. Comparative transcriptome analysis revealed that signaling pathways involving GPCRs and G proteins are dysregulated in the Ric-8B mutant embryos. Interestingly, this analysis also revealed an unexpected impairment of the mTOR signaling pathway. Phosphorylation of Akt at Ser473 is downregulated in the Ric-8B mutant embryos, indicating a decreased activity of mTORC2. Knockdown of the endogenous Ric-8B gene in cultured cell lines leads to reduced phosphorylation levels of Akt (Ser473), further supporting the involvement of Ric-8B in mTORC2 activity. Our results reveal a crucial role for Ric-8B in development and provide novel insights into the signals that regulate mTORC2.

The Chalcone Lonchocarpin Inhibits Wnt/ß-Catenin Signaling and Suppresses Colorectal Cancer Proliferation.

The deregulation of the Wnt/ß-catenin signaling pathway is a central event in colorectal cancer progression, thus a promising target for drug development. Many natural compounds, such as flavonoids, have been described as Wnt/ß-catenin inhibitors and consequently modulate important biological processes like inflammation, redox balance, cancer promotion and progress, as well as cancer cell death. In this context, we identified the chalcone lonchocarpin isolated from Lonchocarpus sericeus as a Wnt/ß-catenin pathway inhibitor, both in vitro and in vivo. Lonchocarpin impairs ß-catenin nuclear localization and also inhibits the constitutively active form of TCF4, dnTCF4-VP16. Xenopus laevis embryology assays suggest that lonchocarpin acts at the transcriptional level. Additionally, we described lonchocarpin inhibitory effects on cell migration and cell proliferation on HCT116, SW480, and DLD-1 colorectal cancer cell lines, without any detectable effects on the non-tumoral intestinal cell line IEC-6. Moreover, lonchocarpin reduces tumor proliferation on the colorectal cancer AOM/DSS mice model. Taken together, our results support lonchocarpin as a novel Wnt/ß-catenin inhibitor compound that impairs colorectal cancer cell growth in vitro and in vivo.

Cholesterol-rich membrane microdomains modulate Wnt/ß-catenin morphogen gradient during Xenopus development.

Wnt/ß-catenin has been described as crucial for dorsal-ventral and antero-posterior patterning, playing multiple roles at different stages of development. Cholesterol-rich membrane microdomains (CRMMs), cholesterol- and sphingolipid-enriched domains of the plasma membrane, are known as platforms for signaling pathways. Although we have demonstrated the importance of the CRMMs for head development, how they participate in prechordal plate formation and embryo axis patterning remains an open question. Moreover, the participation of the CRMMs in the Wnt/ß-catenin signaling pathway activity in vivo is unclear, particularly during embryonic development. In this study, we demonstrated that CRMMs disruption by methyl-beta-cyclodextrin (MßCD) potentiates the activation of the Wnt/ß-catenin signaling pathway in vitro and in vivo during embryonic development, causing head defects by expanding the Wnt expression domain. Furthermore, we also found that the action of CRMMs depends on the microenvironmental context because it also works in conjunction with dkk1, when dkk1 is overexpressed. Thus, we propose CRMMs as a further mechanism of prechordal plate protection against the Wnt signals secreted by posterolateral cells, complementing the action of secreted antagonists.

Developmental aspects of the direct-developing frog Adelophryne maranguapensis.

Direct development in amphibians is characterized by the loss of aquatic breeding. The anuran Adelophryne maranguapensis is one example of a species with direct development, and it is endemic to the state of Ceará, Brazil. Detailed morphological features of A. maranguapensis embryos and the stages of sequential development have not been described before. Here, we analyzed all available genetic sequence tags in A. maranguapensis (tyr exon 1, pomc and rag1) and compared them with sequences from other species of Adelophryne frogs. We describe the A. maranguapensis reproductive tract and embryonic body development, with a focus on the limbs, tail, ciliated cells of the skin, and the egg tooth, which were analyzed using scanning electron microscopy. Histological analyses revealed ovaries containing oocytes surrounded by follicular cells, displaying large nuclei with nucleoli inside. Early in development, the body is unpigmented, and the neural tube forms dorsally to the yolk vesicle, typical of a direct-developing frog embryo. The hindlimbs develop earlier than the forelimbs. Ciliated cells are abundant during the early stages of skin development and are less common during later stages. The egg tooth appears in the later stages and develops as a keratinized microridge structure. The developmental profile of A. maranguapensis presented here will contribute to our understanding of the direct-development model and may help preserve this endangered native Brazilian frog. genesis 54:257-271, 2016. © 2016 Wiley Periodicals, Inc.

Connective-Tissue Growth Factor (CTGF/CCN2) Induces Astrogenesis and Fibronectin Expression of Embryonic Neural Cells In Vitro.

Connective-tissue growth factor (CTGF) is a modular secreted protein implicated in multiple cellular events such as chondrogenesis, skeletogenesis, angiogenesis and wound healing. CTGF contains four different structural modules. This modular organization is characteristic of members of the CCN family. The acronym was derived from the first three members discovered, cysteine-rich 61 (CYR61), CTGF and nephroblastoma overexpressed (NOV). CTGF is implicated as a mediator of important cell processes such as adhesion, migration, proliferation and differentiation. Extensive data have shown that CTGF interacts particularly with the TGFß, WNT and MAPK signaling pathways. The capacity of CTGF to interact with different growth factors lends it an important role during early and late development, especially in the anterior region of the embryo. ctgf knockout mice have several cranio-facial defects, and the skeletal system is also greatly affected due to an impairment of the vascular-system development during chondrogenesis. This study, for the first time, indicated that CTGF is a potent inductor of gliogenesis during development. Our results showed that in vitro addition of recombinant CTGF protein to an embryonic mouse neural precursor cell culture increased the number of GFAP- and GFAP/Nestin-positive cells. Surprisingly, CTGF also increased the number of Sox2-positive cells. Moreover, this induction seemed not to involve cell proliferation. In addition, exogenous CTGF activated p44/42 but not p38 or JNK MAPK signaling, and increased the expression and deposition of the fibronectin extracellular matrix protein. Finally, CTGF was also able to induce GFAP as well as Nestin expression in a human malignant glioma stem cell line, suggesting a possible role in the differentiation process of gliomas. These results implicate ctgf as a key gene for astrogenesis during development, and suggest that its mechanism may involve activation of p44/42 MAPK signaling. Additionally, CTGF-induced differentiation of glioblastoma stem cells into a less-tumorigenic state could increase the chances of successful intervention, since differentiated cells are more vulnerable to cancer treatments.

Derricin and derricidin inhibit Wnt/ß-catenin signaling and suppress colon cancer cell growth in vitro.

Overactivation of the Wnt/ß-catenin pathway in adult tissues has been implicated in many diseases, such as colorectal cancer. Finding chemical substances that can prevent this phenomenon is an emerging problem. Recently, several natural compounds have been described as Wnt/ß-catenin inhibitors and might be promising agents for the control of carcinogenesis. Here, we describe two natural substances, derricin and derricidin, belonging to the chalcone subclass, that show potent transcriptional inhibition of the Wnt/ß-catenin pathway. Both chalcones are able to affect the cell distribution of ß-catenin, and inhibit Wnt-specific reporter activity in HCT116 cells and in Xenopus embryos. Derricin and derricidin also strongly inhibited canonical Wnt activity in vitro, and rescued the Wnt-induced double axis phenotype in Xenopus embryos. As a consequence of Wnt/ß-catenin inhibition, derricin and derricidin treatments reduce cell viability and lead to cell cycle arrest in colorectal cancer cell lines. Taken together, our results strongly support these chalcones as novel negative modulators of the Wnt/ß-catenin pathway and colon cancer cell growth in vitro.

Isoquercitrin suppresses colon cancer cell growth in vitro by targeting the Wnt/ß-catenin signaling pathway.

Flavonoids are plant-derived polyphenolic molecules that have potential biological effects including anti-oxidative, anti-inflammatory, anti-viral, and anti-tumoral effects. These effects are related to the ability of flavonoids to modulate signaling pathways, such as the canonical Wnt signaling pathway. This pathway controls many aspects of embryonic development and tissue maintenance and has been found to be deregulated in a range of human cancers. We performed several in vivo assays in Xenopus embryos, a functional model of canonical Wnt signaling studies, and also used in vitro models, to investigate whether isoquercitrin affects Wnt/ß-catenin signaling. Our data provide strong support for an inhibitory effect of isoquercitrin on Wnt/ß-catenin, where the flavonoid acts downstream of ß-catenin translocation to the nuclei. Isoquercitrin affects Xenopus axis establishment, reverses double axes and the LiCl hyperdorsalization phenotype, and reduces Xnr3 expression. In addition, this flavonoid shows anti-tumoral effects on colon cancer cells (SW480, DLD-1, and HCT116), whereas exerting no significant effect on non-tumor colon cell (IEC-18), suggesting a specific effect in tumor cells in vitro. Taken together, our data indicate that isoquercitrin is an inhibitor of Wnt/ß-catenin and should be further investigated as a potential novel anti-tumoral agent.

Expression and evolution of the Tiki1 and Tiki2 genes in vertebrates.

Tiki1 is a Wnt protease and antagonist specifically expressed in the Spemann-Mangold Organizer and is required for head formation in Xenopus embryos. Here we report neighbor-joining phylogenetic analysis of vertebrate Tiki genes and their mRNA expression patterns in chick, mouse, and rabbit embryos. Tiki1 and Tiki2 orthologues are highly conserved, and exhibit similar but also different developmental expression patterns among the vertebrate/mammalian species analyzed. The Tiki1 gene is noticeably absent in the rodent lineage, but is present in lagomorphs and all other vertebrate/mammalian species examined. Expression in Hensen's node, the equivalent of the Xenopus Organizer, was observed for Chick Tiki2 and Rabbit Tiki1 and Tiki2. Mouse Tiki2 was detected at low levels at gastrulation and head fold stages, but not in the node. Mouse Tiki2 and chick Tiki1 display similar expression in the dorsal spinal cord. Chick Tiki1 expression was also detected in the surface ectoderm and maxillary bud, while chick Tiki2 was found in the anterior intestinal portal, head mesenchyme and primitive atrium. Our expression analyses provide evidence that Tiki1 and Tiki2 are evolutionarily conserved among vertebrate species and their expression in the Organizer and other regions suggests contributions of these Wnt inhibitors to embryonic patterning, as well as organogenesis. Our analyses further reveal mis-regulation of TIKI1 and TIKI2 in human cancer and diseases.

Effects of natural compounds on Xenopus embryogenesis: a potential read out for functional drug discovery targeting Wnt/ß-catenin signaling.

Maternal Wnt/ß-Catenin signaling is essential to establish dorsal-specific gene expression required for axial patterning in Xenopus. Deregulation of this pathway causes axis phenotypes in frog embryos. In adult life, mutations in the Wnt pathway components are associated with many diseases, such as polyposis coli; osteoporosis-pseudoglioma syndrome (OPPG); skeletal dysplasia; neural tube defects, cancer and many others. Thus, a better understanding of Wnt/ß-catenin signaling will have great and significant impact on Biology and Medicine. In this aspect, natural compounds are potential targets as novel molecules that could modulate the Wnt pathway. For instance, flavonoids are a large group of natural compounds found in plants that modulate important cellular and molecular mechanisms related to diseases, but the specific in vivo mechanism of action of most flavonoids remain unknown. In this way, Xenopus embryos may provide an efficient model, since it is frequently used to test and identify the role of molecules that affect Wnt/ß-catenin signaling. Here, we describe a combination of approaches to outline and characterize the role of two flavonoids, quercetin and rutin, on Wnt/ß-catenin signaling, using Xenopus embryos as an experimental model. Our data support that quercetin is potential in vivo modulator of canonical Wnt signaling and that this effect might depend on the structure of this molecule, as we did not observe any effect with rutin treatment, a flavonol structurally-related to quercetin. This model is useful to analyze effects of quercetin and other flavonoids in vivo and to provide further understanding of how natural compounds can modulate signaling pathways, using Xenopus embryos as a fast and efficient reading of in vivo effects of those compounds.

Plasma membrane cholesterol depletion disrupts prechordal plate and affects early forebrain patterning.

Cholesterol-rich membrane microdomains (CRMMs) are specialized structures that have recently gained much attention in cell biology because of their involvement in cell signaling and trafficking. However, few investigations, particularly those addressing embryonic development, have succeeded in manipulating and observing CRMMs in living cells. In this study, we performed a detailed characterization of the CRMMs lipid composition during early frog development. Our data showed that disruption of CRMMs through methyl-ß-cyclodextrin (MßCD) cholesterol depletion at the blastula stage did not affect Spemann's organizer gene expression and inductive properties, but impaired correct head development in frog and chick embryos by affecting the prechordal plate gene expression and cellular morphology. The MßCD anterior defect phenotype was recapitulated in head anlagen (HA) explant cultures. Culture of animal cap expressing Dkk1 combined with MßCD-HA generated a head containing eyes and cement gland. Together, these data show that during Xenopus blastula and gastrula stages, CRMMs have a very dynamic lipid composition and provide evidence that the secreted Wnt antagonist Dkk1 can partially rescue anterior structures in cholesterol-depleted head anlagen.

Dynamic analysis of the expression of the TGFbeta/SMAD2 pathway and CCN2/CTGF during early steps of tooth development.

BACKGROUND/AIMS: CCN2 is present during tooth development. However, the relationship between CCN2 and the transforming growth factor beta (TGFbeta)/SMAD2/3 signaling cascade during early stages of tooth development is unclear. Here, we compare the expression of CCN2 and TGFbeta/SMAD2/3 components during tooth development, and analyze the functioning of TGFbeta/SMAD2/3 in wild-type (WT) and Ccn2 null (Ccn2-/-) mice. METHODS: Coronal sections of mice on embryonic day (E)11.5, E12.5, E13.5, E14.5 and E18.5 from WT and Ccn2-/- were immunoreacted to detect CCN2 and components of the TGFbeta signaling pathway and assayed for 5'-bromo-2'-deoxyuridine immunolabeling and proliferating cell nuclear antigen immunostaining. RESULTS: CCN2 and TGFbeta signaling components such as TGFbeta1, TGFbeta receptor II, SMADs2/3 and SMAD4 were expressed in inducer tissues during early stages of tooth development. Proliferation analysis in these areas showed that epithelial cells proliferate less than mesenchymal cells from E11.5 to E13.5, while at E14.5 they proliferate more than mesenchymal cells. We did not find a correlation between functioning of the TGFbeta1 cascade and CCN2 expression because Ccn2-/- mice showed neither a reduction in SMAD2 phosphorylation nor a difference in cell proliferation. CONCLUSION: CCN2 and the TGFbeta/SMAD2/3 signaling pathway are active in signaling centers of tooth development where proliferation is dynamic, but these mechanisms may act independently.